Process for producing hydroxylated cholesterols and dihydroxycholesterols using amycolata

ABSTRACT

A method for preparing at least one hydroxycholesterol chosen from the group consisting of 25-hydroxycholesterol, 17,25-dihydroxycholesterol and 25,26-dihydroxycholesterol by biological hydroxylation of cholesterol, and the aforementioned dihydroxycholesterols. In the above biological hydroxylation, a microorganism is used which has the abovementioned hydroxylation capacity and which belongs to the genus Amycolata and the genus Sphingomonas.

This application was filed under 35 USC 371 as the national phase ofPCT/JP96/02369 filed Aug. 26, 1996.

TECHNICAL FIELD

The present invention relates to a method for hydroxylating cholesterolby the action of microorganisms, and more specifically to a method forpreparing one or more of either 25-hydroxycholesterol,17,25-dihydroxycholesterol or 25,26-dihydroxycholesterol fromcholesterol. The invention also relates to the aforementioned newdihydroxycholesterols.

BACKGROUND ART

For a biological method, in particular, a method for preparinghydroxy-derivatives of steroids including cholesterol by means ofmicroorganisms, a method by which cholesterol is converted into25-hydroxycholesterol using microorganisms of the genus Streptomyces hasbeen disclosed in Japanese Laid-Open Patent Publication No. 123997/95.Also, in the biological conversion, which is interesting, of compoundsother than steroids, there are known the methods for preparing25-hydroxyvitamin D compounds by the hydroxylation of vitamin Dcompounds using microorganisms, for example, Nocardia autotrophica,Streptomyces roseosporus, Amycolata saturnea, Amycolata autotrophica,Sphingomonas sp. (Japanese Laid-Open Patent Publication Nos. 166090/92,241197/95).

It is known that cholesterol may be, for example, an intermediate on thechemical synthesis of various kinds of vitamin D compounds (Yuki GoseiKagaku (Organic Synthetwic Chemistry) 37, 809-829 (1979)), and compoundswherein one or more of the specific sites of cholesterol is/arehydroxylated beforehand may be expected for their use as intermediateson the synthesis of various hydroxylated vitamin D compounds. Indeed,according to the conversion method using microorganisms described in theabovementioned Japanese Laid-Open Patent Publication No. 123997/95, ithas been published that cholesterol can be selectively hydroxylated atthe 25-position which is preferable in relation to activated vitamin D.

However, the hydroxylation efficiency, according to the method describedin said patent publication, is not always satisfactory. From the viewpoint of improving the water-solubility of final compounds derived fromcholesterol, for example, vitamin D compounds, it would be also desiredto provide not only mono-hydroxylated cholesterols but also furtherhydroxylated cholesterols, for example, dihydroxylated cholesterols.

The purpose of the present invention is therefore to provide a methodfor efficiently preparing mono-hydroxylated cholesterols, in particular25-hydroxycholesterol, and a method for preparing further hydroxylateddihydroxycholesterols, and new dihydroxycholesterols per se.

DISCLOSURE OF INVENTION

In the course of intensive study for the accomplishment of the abovepurpose, the present inventors have found that microorganisms of generaother than the genus Streptomyces described in the abovementionedJapanese Laid-Open Patent Publication No. 123997/95 hydroxylatecholesterol not only at the 25-position but also at the 17- or26-position.

Thus, the above purpose can be achieved by providing a method, by whichcholesterol having formula (I) ##STR1## is biologically converted intohydroxylated cholesterols of formula (II) ##STR2## (in which R¹ is ahydroxyl radical, and R² and R³ are a hydroxyl radical and a hydrogenatom, respectively, or a hydrogen atom and a hydroxyl radical,respectively), for preparing hydroxylated cholesterols having theformula (II), according to the invention, comprising

(A) a step in which the aforementioned biological conversion can becarried out, and in which cholesterol having the formula (I) is treatedby incubation in the presence of a microorganism, chosen from those thatbelong to the genus Amycolata and the genus Sphingomonas, or itspreparation from cultures and oxygen, and

(B) a step in which at least one of the hydroxylcholesterols having theformula (II) is recovered from the incubation-treated solution.

Among the hydroxylated cholesterols having the formula (II),dihydroxycholesterols of formula (II-b) ##STR3## (in which R² and R³ area hydroxyl radical and a hydrogen atom, respectively, or a hydrogen atomand a hydroxyl radical, respectively) can be also prepared by anothermethod in which 25-hydroxycholesterol is substituted for cholesterol asa starting material in the above method.

Furthermore, dihydroxycholesterols having the above formula (II-b), thatis, 17,25-dihydroxycholesterol and 25,26-dihydroxycholesterol, arecompounds that have not been published in literature in the prior art.Therefore, according to the present invention, new compounds,dihydroxycholesterols having the formula (II-b), are also provided.

DETAILED DESCRIPTION OF THE INVENTION

In the biological conversion according to the present invention,microorganisms and their preparations from cultures can be used,regardless of the kinds of species and strains, provided that they aremicroorganisms belonging to the genus Amycolata and the genusSphingomonas and having the capacity to convert cholesterol of the aboveformula (I) into hydroxylated cholesterols of the formula (II). Mentioncan be made, as preferred microorganisms, Amycolata saturnea having theabovementioned conversion capacity, in particular, the microorganismsthat have been deposited at the National Institute of Bioscience andHuman Technology, the Agency of Industrial Science and Technology, theMinistry of International Trade and Industry in Japan with thedeposition numbers of FERM BP-5544 and FERM BP-2307, and Amycolataautotrophica, in particular, the strain that has been deposited atAmerican Type Culture Collection in the United States with thedeposition number of ATCC 33796.

Species that belong to the genus Sphingomonas, mali IFO 15500,paucimobilis IFO 13935, parapaucimobilis IFO 15100, yanoikuyae IFO15102, adhaesiva IFO 15099, capsulata IFO 12533, sanguis IFO 13937,macrogoltabidus IFO 15033 and terrae IFO 15098, although they areinferior in conversion capacity compared to that of microorganismsbelonging to the above-described genus Amycolata, can be also used.Here, IFO is a deposition number in the Institute for Fermentation inJapan.

According to the invention, cholesterol (the compound of formula (I))and/or 25-hydroxycholesterol (the compound of formula (II-a)) being astarting material (or a substrate) will be treated by incubation in thepresence of any of said strains or their mycelia from cultures andoxygen. This treatment can be carried out by adding a substrate, at thetime of the cultivation of the above strain under the aerobicconditions, into a culture solution, or optionally by adding a substrateinto a suspension of, for example, the mycelia as such or thehomogenized preparations obtained from cultures of the above strains,followed by incubation with oxygen, for example, with air. The additionof a substrate into a culture solution may be performed either beforethe cultivation or at a certain period of time after the cultivation.The above mycelia can be prepared by inoculating any of the abovestrains into a medium containing nutrient sources, followed by aerobiccultivation.

The cultivation of a strain to obtain such bacterial preparation fromcultures or the cultivation of a strain carried out with the addition ofa substrate can be performed, in principle, in accordance withcultivation methods for general microorganisms, but it is usuallypreferable to be carried out under aerobic conditions such as shakingliquid culture, aerated and stirred culture, etc.

The media used for the cultivation may be those containing nutrientsources which can be utilized by microorganisms belonging to the genusAmycolata and the genus Sphingomonas, and any of various kinds ofsynthetic or semi-synthetic media, natural media and the like can beused. For the medium compositions, glucose, maltose, xylose, fructose,sucrose and the like can be used alone or in combination as carbonsources. For nitrogen sources, organic nitrogen sources such as peptone,meat extract, soybean meal, casein, amino acids, yeast extract, urea andthe like, and inorganic nitrogen sources such as sodium nitrate,ammonium sulfate and the like can be used alone or in combination.Furthermore, if necessary, for example, salts such as sodium chloride,potassium chloride, calcium carbonate, magnesium sulfate, sodiumphosphate, potassium phosphate, cobalt chloride and the like, salts ofheavy metals, vitamins can also be used. In case where foaming isfurious during the cultivation, various known defoamers can be alsoadded suitably into a medium.

Cultivation conditions can be suitably selected so that said strains cangrow well. Usually, the cultivation are performed at pH 6-7.5, at 28-30°C. for approximately 2-8 days. Various cultivation conditions describedabove can be suitably changed depending on the kind and property of amicroorganism used, external conditions and the like, and optimizedconditions can be easily selected by those skilled in the art.

Alternatively, after completion of the cultivation, bacterialpreparation from cultures is prepared by suspending mycelia, which hasbeen separated by centrifugation or filtration, or homogenized myceliain an appropriate solution. Solutions that can be used for suspendingmycelia are the media as described above or buffer solutions such astris-acetate, tris-hydrochloride, sodium succinate, sodium citrate,sodium phosphate, potassium phosphate and the like and they are usedalone or in admixture. For the pH values of the buffer solution,preferably 6.0-9.0 and more preferably 7.0-8.5 can be mentioned.

A substrate can be added into a culture solution or a bacterialsuspension in the form of powder or by dissolving in water-solubleorganic solvent, for example, ethanol and the like, and the amount to beadded, for example, in the case of a culture solution, is preferably0.15-0.60 mg per 1 ml of the culture solution. When the amount isincreased to more than 0.60 mg/ml, conversion rate becomes slow and isnot preferable. After the addition of a substrate, the substrate can beconverted into an objective hydroxylated cholesterol by carrying out theoperation of shaking or aeration-agitation and the like at 27-31° C. for1-3 days, preferably approximately for one day, to allow the reaction toproceed under aerobic conditions. In such a conversion reaction, theconversion rate into an objective hydroxylated cholesterol from asubstrate can be remarkably increased by adding the substrate andmethylated cyclodextrins to a reaction solution.

In a preferred embodiment according to the invention, the incubationtreatment described above is therefore carried out further in thepresence of methylated cyclodextrins.

Methylated cyclodextrins used according to the invention refer tocompounds wherein hydrogen atoms of hydroxyl radicals at the 2-, 3- or6-position of cyclodextrin are substituted by methyl radicals, andhexakis-(2,6-O-dimethyl)-α-cyclodextrin, derived from α-cyclodextrin,heptakis-(2,6-O-dimethyl)-β-cyclodextrin derived from β-cyclodextrin andoctakis-(2,6-O-dimethyl)-γ-cyclodextrin derived from γ-cyclodextrin,which are completely methylated at the 2- and 6-positions, orhexakis-(2,3,6-O-trimethyl)-α-cyclodextrin derived from α-cyclodextrin,heptakis-(2,3,6-O-trimethyl)-β-cyclodextrin derived from β-cyclodextrinand octakis-(2,3,6-O-trimethyl)-γ-cyclodextrin derived fromγ-cyclodextrin, which are completely methylated at the 2-, 3- and6-positions, or partially methylated cyclodextrins wherein each of 6, 7or 8 hydroxyl radicals at the position of the 2-, 3- and 6-positions arepartially methylated can be mentioned. In the present invention, one ormore of any methylated cyclodextrins is/are selected from thosedescribed above and used, but, in particular, partially methylatedcyclodextrin derived from β-cyclodextrin is preferably used.

The amount of methylated cyclodextins added is 0.5 mg or more,preferably 0.5-15 mg, and more preferably 1-10 mg per 1 ml of a reactionsolution. When the amount of the methylated cyclodextrins added is lessthan 0.5 mg per 1 ml of a reaction solution, there are sometimes caseswhere the increase in conversion rate into an objective hydroxylatedcholesterol does not become significant compared to that without theaddition, and for the amount in the region of 15 mg, foaming takes placein some cases, and it may become necessary to use a defoamer etc.together.

In the method according to the invention, nonionic surfactants may beadded into a reaction mixture so as not to decrease the conversion ratedescribed above. Mention can be made, as such a surfactant, ofpolyoxyethylene.sorbitan fatty acid ester (e.g., Tween^(R) 80 (Sigma)),sorbitan fatty acid ester (e.g., Span^(R) 85 (Sigma)), polyoxyethyleneether (e.g., Brij^(R) 96 (Sigma)) and Triton^(R) X-100 (Sigma),nonylphenol (e.g., Nonypol^(R) 45 (Sanyo Chemical Industries Ltd.),block copolymer of ethylene oxide-propylene oxide (e.g., Pluronic^(R)L-61 (Asahi Denka Kogyo K. K.), and Dislex^(R) (Nippon Oil and Fats Co.Ltd.) as an anionic surfactant, Trax^(R) (Nippon Oil and Fats Co. Ltd.)and the like.

To isolate an objective hydroxylated cholesterol thus produced from areaction mixture, various known purification procedures can be selectedand carried out in combination. For example, it can be separated andpurified by means of adsorption to hydrophobic adsorption resins andelusion, extraction with solvent using ethyl acetate, n-butanol etc., acolumn chromatography with silica gel etc. or thin layer chromatography,preparative high performance liquid chromatography using a reversedphase column and the like and these can be used alone or suitably incombination, or optionally used repeatedly.

An Amycolata saturnea FERM BP-5544, which is one of the strains beingable to be used particularly advantageously in the abovementionedbiological conversion, was isolated from soil by the present inventors,and named A-1246 strain, and it is a new strain as described belowshowing bacteriological properties as follows:

(1) Morphology

Vegetative mycelium develops well on synthetic or natural agar media andbranches irregularly. No septum is observed. Spore chains are formedabundantly on glycerin-asparagine agar media, starch-inorganic saltsagar media and the like. By microscopic observation, sporulatingmycelium branches monopodially with straight spore chains. Usually, thespore chains have three or more spores, and the long spore chains aredeveloped at the late growth phase of the culture with smooth surfaces.The spore is cylindrical in shape and 0.5-0.8×2.5-4.3 μm in size.Sclerotia, sporangia and flagellated spore are not observed.

(2) Growth on Various Media (30° C.)

(2-1) Sucrose-nitrate Agar Medium

Growth on the medium is moderate and the color of the reverse side ofcolonies is pale brown. Aerial mycelium forms moderately and colorscreamy. No soluble pigment is produced.

(2-2) Glucose-asparagin Agar Medium

Growth on the medium is slightly poor and the color of the reverse sideof colonies is creamy. Aerial mycelium forms moderately and colorswhite. No soluble pigment is produced.

(2-3) Glycerin-asparagin Agar Medium

Growth on the medium is good and the color of the reverse side ofcolonies is pale yellow. Aerial mycelium forms well and colors white. Nosoluble pigment is produced.

(2-4) Starch-inorganic Salts Agar Medium

Growth on the medium is moderate and the color of the reverse side ofcolonies is creamy. Aerial mycelium forms well and colors white. Nosoluble pigment is produced.

(2-5) Tyrosine Agar Medium

Growth on the medium is moderate and the color of the reverse side ofcolonies is reddish brown. Aerial mycelium forms well and colors creamy.Soluble pigment with pale reddish brown color is produced.

(2-6) Nutrient Agar Medium

Growth on the medium is good and the color of the reverse side ofcolonies is pale yellow. Aerial mycelium forms well and colors white. Nosoluble pigment is produced.

(2-7) Yeast-malt Extract Agar Medium

Growth on the medium is good and the color of the reverse side ofcolonies is pale yellow. Aerial mycelium forms slightly poorly andcolors white. No soluble pigment is produced.

(2-8) Oatmeal Agar Medium

Growth on the medium is moderate and the color of the reverse side ofcolonies is creamy. Aerial mycelium forms slightly poorly and colorswhite. No soluble pigment is produced.

(2-9) Peptone.Yeast.Iron Agar Medium

Growth on the medium is moderate and the color of the reverse side ofcolonies is pale brown. Aerial mycelium forms moderately and colorscreamy. No soluble pigment is produced.

(3) Physiological Properties

(3-1) Temperature Range for Growth

When nutrient agar medium is used, good growth is observed at thetemperature in the range of 20-30° C. There is no growth at 10° C. orbelow, and at 40° C. or above

(3-2) Distinction Between Aerobic and Anaerobic: Aerobic

(3-3) Liquefaction of Gelatin: Positive

(3-3) Hydrolysis of Starch: Negative

(3-4) Coagulation and Peptonization of Skim Milk: Both Negative

(3-5) Formation of Melanin-like Pigment: Negative

(3-6) Nitrate Reduction: Negative

(4) Utilization of Carbon Sources

When a carbon source is added onto Pridham.Godlieb agar medium and thegrowth is observed, any carbon sources of the followings: D-glucose,sucrose, D-xylose, inositol, D-mannitol, D-fructose, can be utilized.L-arabinose, L-rhamnose and raffinose cannot be utilized.

(5) Cell Wall Components

As a result of the analysis of cell wall components with whole bacteriallysate, the cell wall of this strain belongs to the type III accordingto the classification by Lechevalier (International Journal ofSystematic Bacteriology, vol. 20, p435-443 (1970)). Mycolic acid is notcontained.

It is apparent form the above bacteriological properties that thisstrain belongs to Actinomycetes, and when these properties were comparedwith those of known microorganisms reported in the International Journalof Systematic Bacteriology, Vol.36, p29-37 (1986), this strain wasalmost identical to Amycolata saturnea. As a result of the above, thisstrain is concluded to belong to Amycolata saturnea, and named anAmycolata saturnea A-1246 strain. After deposited at the NationalInstitute of Bioscience and Human Technology, the Agency of IndustrialScience and Technology in Japan as FERM P-15098 on Aug. 7, 1995, thisstrain was transferred to the International Depositary Authority in theinstitute and given the deposition number FERM BP-5544 in compliancewith the provisions of the Budapest Convention on InternationalAcknowledgement of the Deposition of Microorganisms for the purpose ofPatent Proceedings.

The present invention is illustrated in more detail by the followingExamples, which are not intended to limit the invention.

Unless mentioned otherwise, the percentages in the examples belowrepresents percent by weight.

EXAMPLES 1-9 Conversion into 25-hydroxycholesterol from Cholesterol

One hundred ml of a seed culture medium consisting of 1.5% of glucose,1.5% of Bacto®-soyton (Difco), 0.5% of corn steep liquor, 0.4% of sodiumchloride and 0.2% of calcium carbonate (pH 7.0) was placed in a 500 mlErlenmeyer flask and autoclaved at 120° C. for 20 minutes. This mediumwas inoculated with 2 ml of a frozen inoculum of an Amycolata saturneaA-1246 strain (FERM BP-5544) and shaking culture was carried out at 220rpm for 48 hours at 28° C., thus a seed culture solution being prepared.

Fifty ml of a conversion culture medium consisting of 2.0% of glucose,0.2% of yeast extract, 0.5% of peptone, 1.0% of soybean meal, 0.5% ofcorn steep liquor, 0.04% of potassium secondary phosphate, 0.04% ofsodium chloride and 0.2% of calcium carbonate (pH 7.4) was then placedin a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20 minutes.This medium was inoculated with 1 ml of the seed culture solutionprepared above and shaking culture was carried out at 220 rpm at 28° C.Fifteen mg of the substrate cholesterol and each compound which is shownin Table 1 were added 48 hours later, and the cultivation continued forfurther 72 hours.

The substrate and the like were added in the following way: Fifteen mgof cholesterol in the form of powder was added in Example 1. For Example2, 150 mg of cholesterol was suspended in 4 ml of ethanol, dispersed bysonication, and 0.4 ml of the suspension was added. In Example 3, 150 mgof cholesterol was suspended in a mixture of 1 ml of Tween 80(surfactant: Sigma) and 3 ml of ethanol, dispersed by sonication, and0.4 ml of the suspension was added. In Examples 4, 5 and 6, there wereadded 15 mg of cholesterol, 1 ml of Tween 80 and 5.6 ml of a 1.5%aqueous solution of each cyclodextrin which was sterilized beforehand(121° C., 20 minutes). In Examples 7, 8 and 9, there were added 15 mg ofcholesterol, 1 ml of Tween 80 and a 1.5% aqueous solution of partiallymethylated β-cyclodextrin (methylation rate 74%: Mercian Corp.), whichwas sterilized beforehand (121° C., 20 minutes), at each determinedconcentration.

Two ml of the culture solution obtained was collected into acentrifugation tube with a stopper, to which 0.5 ml of ethyl acetate wasadded, and stirring for 30 minutes and further centrifugation at 3000rpm for 15 minutes separated off the ethyl acetate layer. Five μl ofthis was spotted onto a TLC plate (Silica gel 60 F₂₅₄ : Merck),developed with chloroform:methanol=10:1 and stained with sulfuric acid.The spot indicating the same Rf value (approximately 0.5) as a standardproduct (Sigma) was scanned by Chromatoscanner (CS-920: Shimadzu Corp.),25-hydroxycholesterol being quantified. The conversion rates into25-hydroxycholesterol from cholesterol were summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                                             Concentration                                              Example Coexisting of coexisting Conversion                                   No. substance substance (%) rate (%)                                        ______________________________________                                        1        --          --         0.8                                             2 Ethanol 0.80 0.7                                                            3 Tween 80 0.20 0.7                                                           4 α-CD 0.15 0.7                                                         5 β-CD 0.15 0.7                                                          6 γ-CD 0.15 0.8                                                         7 β-PMCD 0.05 5.8                                                        8 β-PMCD 0.10 10.3                                                       9 β-PMCD 0.15 12.1                                                     ______________________________________                                         CD: cyclodextrin                                                              CD: cyclodextrin                                                              CD: cyclodextrin                                                              PMCD: partially methylated cyclodextrin                                  

EXAMPLES 10-19 Conversion into 25-hydroxycholesterol from Cholesterol(Effect of Alteration in the Reaction Time)

Fifty ml of the conversion culture medium described in Examples 1-9 wasplaced in a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20minutes. This medium was inoculated with 1 ml of the seed culturesolution prepared as in Examples 1-9 and shaking culture was carried outat 220 rpm for 48 hours at 28° C. To this culture, 15 mg of cholesterolin the form of powder and 1 ml of Tween 80 were added, a 1.5% aqueoussolution of each methylated β-cyclodextrin which was sterilizedbeforehand was further added so as to be a final concentration of 0.15%,and the cultivation continued. At 16, 40 and 64 hours after the additionof cholesterol, 25-hydroxycholesterol in the culture solution wasquantified by the same method as Examples 1-9. The conversion rates into25-hydroxycholesterol from cholesterol were summarized in Table 2. InExamples 17 and 18, the mixtures of 2,6-di-O-methyl-β-cyclodextrin and2,4,6-tri-O-methyl-β-cyclodextrin were used at the ratios of 2:1 and1:2, respectively.

                  TABLE 2                                                         ______________________________________                                        Example  Coexisting   Methylation                                                                             Conversion rate (%)                           No.      substance    rate (%)  16 hr                                                                              40 hr                                                                              64 hr                               ______________________________________                                        10       --           --         0.0 0.5  0.8                                   11 β-PMCD 56 11.6 7.6 10.5                                               12 β-PMCD 62 13.1 10.3  9.6                                              13 β-PMCD 68 11.3 10.4  10.7                                             14 β-PMCD 69  8.1 6.2 6.9                                                15 β-PMCD 74 13.5 7.6 10.5                                               16 β-DMCD 67 10.8 8.5 7.9                                                17 β-DMCD + TMCD 78 17.1 14.0  16.0                                      18 β-DMCD + TMCD 89 11.6 11.4  --                                        19 β-TMCD 100   0.0 2.9 4.3                                            ______________________________________                                         PMCD: partially methylated cyclodextrin                                       DMCD: 2,6di-O-methyl-cyclodextrin                                             TMCD: 2,4,6tri-O-methyl-cyclodextrin                                     

EXAMPLES 20-23 Conversion into 25-hydroxycholesterol from Cholesterol(Effect of Alteration in the Concentration of Cholesterol Added)

Fifty ml of the conversion culture medium described in Examples 1-9 wasplaced in a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20minutes. This medium was inoculated with 1 ml of the seed culturesolution prepared as in Examples 1-9 and shaking culture was carried outat 220 rpm for 48 hours at 28° C. To this culture were added cholesterolin the form of powder and 5.6 ml of a 1.5% aqueous solution of partiallymethylated β-cyclodextrin (methylation rate 74%: Mercian Corp.) whichwas sterilized beforehand, and the cultivation continued for further 40hours. By the same method as Examples 1-9, 25-hydroxycholesterol in theculture solution obtained was quantified. The conversion rates into25-hydroxycholesterol from cholesterol were summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                        Example                                                                              Concentration                                                                              Concentration of                                                                             Conversion                                   No. of CHO added 25OH--CHO produced rate (%)                                ______________________________________                                        20     0.15 mg/ml   32 μg/ml    21.3                                         21 0.30 mg/ml 55 μg/ml 18.3                                                22 0.60 mg/ml 34 μg/ml  5.6                                                23 1.20 mg/ml  5 μg/ml  0.4                                              ______________________________________                                         CHO: Cholesterol                                                              25OH--CHO: 25hydroxycholesterol                                          

EXAMPLES 24-26 Conversion into 25-hydroxycholesterol from Cholesterol(by Bacterial Preparations)

Fifty ml of the conversion culture medium described in Examples 1-9 wasplaced in a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20minutes. This medium was inoculated with 1 ml of the seed culturesolution prepared as in Examples 1-9 and shaking culture was carried outat 220 rpm for 48 hours at 28° C. The resulting culture solution wascentrifuged (3000 rpm, 10 minutes) to collect the mycelia, which wasthen suspended in 50 ml of each buffer solution having the followingcompositions.

Buffer Solution A (Example 24)

50 mM tris-acetate, 25 mM sodium succinate, 0.05% magnesium sulfate,0.15% partially methylated β-cyclodextrin (methylation rate 74%: MercianCorp.), 0.5% glucose (pH 8.0)

Buffer Solution B (Example 25)

50 mM tris-acetate, 25 mM sodium succinate, 0.05% magnesium sulfate,0.15% partially methylated β-cyclodextrin (methylation rate 74%: MercianCorp.) (pH 8.0)

Buffer Solution C (Example 26)

50 mM tris-acetate, 25 mM sodium succinate, 0.05% magnesium sulfate (pH8.0)

Fifteen mg of cholesterol in the form of powder was added into each ofthese suspensions, and the mixture was incubated with shaking at 220 rpmfor 24 hours at 28° C. By the same method as Examples 1-9,25-hydroxycholesterol in the resulting solution was quantified. Theconversion rates into 25-hydroxycholesterol from cholesterol weresummarized in Table 4.

                  TABLE 4                                                         ______________________________________                                        Example No. Buffer solution                                                                           Conversion rate (%)                                   ______________________________________                                        24          Buffer solution A                                                                         13.8                                                    25 Buffer solution B 15.4                                                     26 Buffer solution C  0.5                                                   ______________________________________                                    

EXAMPLE 27 Conversion into 25-hydroxycholesterol from Cholesterol (ScaleUp)

One hundred ml of a seed culture medium consisting of 1.5% of glucose,1.5% of Bacto®-soyton (Difco), 0.5% of corn steep liquor, 0.4% of sodiumchloride and 0.2% of calcium carbonate (pH 7.0) was placed in a 500 mlErlenmeyer flask and autoclaved at 120° C. for 20 minutes. This mediumwas inoculated with 2 ml of a frozen inoculum of an Amycolata saturneaA-1246 strain (FERM BP-5544) and shaking culture was carried out at 220rpm for 48 hours at 28° C., thus a seed culture solution being prepared.

Then, 1.5 l of a conversion culture medium consisting of 2.0% ofglucose, 0.2% of yeast extract, 0.5% of peptone, 1.0% of soybean meal,0.5% of corn steep liquor, 0.04% of potassium secondary phosphate, 0.04%of sodium chloride, 0.2% of calcium carbonate and 0.05% of Silicon KM75(defoamer: Shin-Etsu Chemical Co., Ltd.) (pH 7.4) was placed in each offive 3 l mini-jar and sterilized by heating at 120° C. for 20 minutes.Each medium was inoculated with 30 ml of the seed culture solutionprepared above and cultivation was carried out for 48 hours at thetemperature of 28° C., at the agitation of 400 rpm and at the aerationof 1.0 vvm. To this culture, 450 mg of cholesterol dissolved in 15 ml ofethanol was added, 4 ml of a 10% aqueous solution of Silicon KM75(defoamer) and 30 ml of a 7.5% aqueous solution of partially methylatedβ-cyclodextrin (methylation rate 74%: Mercian Corp.) were further added,and the cultivation continued for further 72 hours. The concentration of25-hydroxycholesterol quantified by the same method as Example 1 was 114μg/ml at 24 hours, 62 μg/ml at 48 hours, and 49 μg/ml at 72 hours afterthe addition of cholesterol. Thus, 5.6 l of combined culture solutionwas obtained from five mini-jars.

Following the addition of Pearlite (filter aid: Toko Pearlite IND.) at aconcentration of 3%, this culture solution was filtrated to obtainfiltrate. Five l of ethyl acetate and 500 g of sodium chloride wereadded and the mixture was stirred for 90 minutes by a stirrer. Twohundreds ml of ethanol was further added and the mixture allowed tostand for 1 hour. After the ethyl acetate layer (about 4.5 l) wasseparated off and concentrated under reduced pressure to a volume ofapproximately 50 ml, 50 ml of ethyl acetate, 5 g of sodium chloride and2 ml of ethanol were further added to extract the product into theorganic layer. After the ethyl acetate layer (about 50 ml) was separatedoff and concentrated under reduced pressure to a volume of approximately4 ml, 50 ml of deionized water was added. The resulting suspension wasextracted twice with 30 ml of chloroform. The chloroform layer was driedover anhydrous sodium sulfate and concentrated to dryness, giving 800 mgof yellow powder.

The resulting powder was dissolved in the lower layer ofchloroform-methanol-water (7:13:8) and purified by centrifugedliquid-liquid partition chromatography (CPC model NMF, 250W×12: SankiEngineering Co., Ltd.) using this two-layer system: Portions where aspot was observed at the Rf value of about 0.5 in the TLC analysis as inExample 1 were collected and concentrated to dryness, giving 168.2 mg ofwhite powder. This powder was further subjected to column chromatographyon Sephadex^(R) LH-20 (Pharmacia, developing solvent: toluene-methanol(85:15)) and fractions that gave single spot in the above TLC analysiswere combined and concentrated to dryness, giving 133.5 mg of25-hydroxycholesterol in the form of white powder.

The resulting powder was dissolved in methanol at a concentration of 0.1mg/ml and subjected to a HPLC analysis under the following conditions,single peak with the retention time of about 17.9 minutes beingobserved. This retention time was in agreement with that of a standardproduct of 25-hydroxycholesterol (Sigma).

(HPLC Conditions)

Column: YMC pack A-302 (φ 4.6 mm×150 mm)

Mobile phase: 85% methanol

Flow rate: 0.8 ml/minute

Detection: 210 nm

Mass spectrum and NMR spectrum of the resulting powder were also inagreement with those of the standard product.

(1) FAB mass spectrum (positive ion, matrix NBA): m/z=402 (M⁺)

(2) ¹ H-NMR spectrum (CDCl₃ : internal standard TMS): 0.68 (3H, s), 0.93(3H, d, J=6.2 Hz), 1.01 (3H, s), 1.22 (6H, s), 3.53 (1H, m), 5.35 (1H,m)

EXAMPLE 28 Conversion into 17,25-dihydroxycholesterol from Cholesterol

A seed culture solution was prepared as in Examples 1-9.

Then, 1.5 l of a conversion culture medium consisting of 2.0% ofglucose, 0.2% of yeast extract, 0.5% of peptone, 1.0% of soybean meal,0.5% of corn steep liquor, 0.04% of potassium secondary phosphate, 0.04%of sodium chloride, 0.2% of calcium carbonate and 0.05% of Silicon KM75(defoamer: Shin-Etsu Chemical Co., Ltd.) (pH 7.4) was placed in a 3 lmini-jar and sterilized by heating at 120° C. for 20 minutes. Thismedium was inoculated with 30 ml of the seed culture solution preparedabove and cultivation was carried out for 48 hours at the temperature of28° C., at the agitation of 400 rpm and at the aeration of 1.0 vvm. Tothis culture, 450 mg of cholesterol dissolved in 15 ml of ethanol wasadded, 4 ml of a 10% aqueous solution of Silicon KM75 (defoamer:Shin-Etsu Chemical Co., Ltd.) and 30 ml of a 7.5% aqueous solution ofpartially methylated β-cyclodextrin (methylation rate 74%: MericanCorp.) were further added, and the cultivation continued for further 91hours.

Following the addition of Pearlite (filter aid: Toko Pearlite IND.) at aconcentration of 3%, the culture solution thus obtained was filtrated togive 1.2 l of filtrate. The filtrate was passed through a 100 mlAmberlite XAD-8 (Rohm & Haas) column to adsorb the product. Afterwashing this column with 200 ml of 20% methanol, the product was elutedwith 500 ml of 90% aqueous methanol. After concentrating the eluateunder reduced pressure to remove methanol, about 100 ml of the residualwater layer was extracted twice with 50 ml of ethyl acetate. Theresulting ethyl acetate layer was dried over anhydrous sodium sulfateand concentrated to dryness.

After applying this residue to a 100 ml silica gel (trade name: Silicagel 60, Merck) column, eluting with 400 ml of chloroform-methanol(100:1) and concentrating the eluate to dryness to remove the solvent,the residue was further subjected to preparative thin layerchromatography (trade name: Silica gel 60 Art11798, Merck) and developedwith chloroform-methanol (20:1). The portion corresponding to17,25-dihydroxycholesterol (Rf=0.33) was scraped off, extracted withchloroform-methanol (1:1) and concentrated to dryness, giving 3.3 mg of17,25-dihydroxycholesterol in the form of white powder.

The physical and chemical properties of 17,25-dihydroxycholesterolprovided according to the present invention were shown below:

(1) Appearance: white powder

(2) Molecular formula: C₂₇ H₄₆ O₃

(3) FAB mass spectrum (negative ion, matrix NBA): m/z=417 (M-1)

(4) ¹ H-NMR spectrum (400 MHZ, CDCl₃): Main absorptions were as follows:

δ TMS (ppm): 0.78 (3H, s), 0.92 (3H, d, J=6.2 Hz), 1.00 (3H, s), 1.22(6H, s), 3.53 (1H, m), 5.36 (1H, m)

(5) ¹³ C-NMR spectrum (100 MHz, CDCl₃): Main absorptions were asfollows:

δ TMS (ppm): 14.0 (q), 14.4 (q), 19.4 (q), 20.9 (t), 22.6 (t), 23.7 (t),29.3 (q×2), 31.7 (t), 31.9(t), 32.2 (d), 32.3 (t), 32.7 (t), 36.5 (s),37.3 (t), 38.1 (t), 39.7 (d), 42.3 (t), 44.2 (t), 47.3 (s), 49.7 (d),51.2 (d), 71.0 (s), 71.8 (d), 86.5 (s), 121.7 (d), 140.7 (s)

EXAMPLES 29-37 Preparation of 17,25-dihydroxycholesterol (Effect ofAlteration in the Coexisting Substance on Conversion Rate)

One hundred ml of a seed culture medium consisting of 1.5% of glucose,1.5% of Bacto®-soyton (Difco), 0.5% of corn steep liquor, 0.4% of sodiumchloride and 0.2% of calcium carbonate (pH 7.0) was placed in a 500 mlErlenmeyer flask and autoclaved at 120° C. for 20 minutes. This mediumwas inoculated with 2 ml of a frozen inoculum of an Amycolata saturneaA-1246 strain (FERM BP-5544) and shaking culture was carried out at 220rpm for 48 hours at 28° C., thus seed culture solution being prepared.

Fifty ml of a conversion culture medium consisting of 2.0% of glucose,0.2% of yeast extract, 0.5% of peptone, 1.0% of soybean meal, 0.5% ofcorn steep liquor, 0.04% of potassium secondary phosphate, 0.04% ofsodium chloride and 0.2% of calcium carbonate (pH 7.4) was then placedin a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20 minutes.This medium was inoculated with 1 ml of the seed culture solutionprepared above and shaking culture was carried out at 220 rpm at 28° C.Fifteen mg of the substrate cholesterol and each compound which is shownin Table 5 were added 48 hours later, and the cultivation continued forfurther 72 hours.

The substrate and the like were added in the following way: Fifteen mgof cholesterol in the form of powder was added in Example 29. ForExample 30, 150 mg of cholesterol was suspended in 4 ml of ethanol,dispersed by sonication, and 0.4 ml of the suspension was added. InExample 31, 150 mg of cholesterol was suspended in a mixture of 1 ml ofTween 80 (surfactant: Sigma) and 3 ml of ethanol, dispersed bysonication, and 0.4 ml of the suspension was added. In Examples 32, 33and 34, there were added 15 mg of cholesterol, 1 ml of Tween 80 and 5.6ml of a 1.5% aqueous solution of each cyclodextrin which was sterilizedbeforehand (121° C., 20 minutes). In Examples 35, 36 and 37, there wereadded 15 mg of cholesterol, 1 ml of Tween 80 and a 1.5% aqueous solutionof partially methylated β-cyclodextrin (methylation rate 74%: MercianCorp.), which was sterilized beforehand (121° C., 20 minutes), at eachdetermined concentration.

Two ml of the culture solution obtained was collected into acentrifugation tube with a stopper, to which 0.5 ml of ethyl acetate wasadded, and stirring for 30 minutes and further centrifugation at 3000rpm for 15 minutes separated off the ethyl acetate layer. Five μl ofthis was spotted onto a TLC plate (Silica gel 60 F₂₅₄ : Merck),developed with chloroform:methanol=10:1 and stained with sulfuric acid.The spot indicating the same Rf value (0.33) as the standard product inExample 1 was scanned by Chromatoscanner (CS-920: Shimadzu Corp.),17,25-hydroxycholesterol being quantified. The conversion rates into17,25-dihydroxycholesterol from cholesterol were summarized in Table 5.

                  TABLE 5                                                         ______________________________________                                                             Concentration                                              Example Coexisting of coexisting Conversion                                   No. substance substance (%) rate (%)                                        ______________________________________                                        29       --          --         0.3                                             30 Ethanol 0.80 0.2                                                           31 Tween 80 0.20 0.2                                                          32 α-CD 0.15 0.3                                                        33 β-CD 0.15 0.2                                                         34 γ-CD 0.15 0.3                                                        35 β-PMCD 0.05 2.0                                                       36 β-PMCD 0.10 2.8                                                       37 β-PMCD 0.15 3.5                                                     ______________________________________                                         CD: cyclodextrin                                                              CD: cyclodextrin                                                              CD: cyclodextrin                                                              PMCD: partially methylated cyclodextrin                                  

EXAMPLES 38-47 Preparation of 17,25-dihydroxycholesterol (Effect ofAlteration in the Reaction Time)

Fifty ml of the conversion culture medium described in Examples 29-37was placed in a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20minutes. this medium was inoculated with 1 ml of the seed culturesolution prepared as in Example 2 and shaking culture was carried out at220 rpm for 48 hours at 28° C. Fifteen mg of cholesterol in the form ofpowder and 1 ml of Tween 80 were added to this culture, a 1.5% aqueoussolution of each methylated β-cyclodextrin which was sterilizedbeforehand was further added so as to be a final concentration of 0.15%,and the cultivation continued. At 16, 40 and 64 hours after the additionof cholesterol, 17,25-hydroxycholesterol in the culture solution wasquantified by the same method as Examples 29-37. The conversion ratesinto 17,25-dihydroxycholesterol from cholesterol were summarized inTable 6. In Examples 45 and 46, the mixtures of2,6-di-O-methyl-β-cyclodextrin and 2,4,6-tri-O-methyl-β-cyclodextrinwere used at the ratios of 2:1 and 1:2, respectively.

                  TABLE 6                                                         ______________________________________                                        Example  Coexisting   Methylation                                                                             Conversion rate (%)                           No.      substance    rate (%)  16 hr                                                                              40 hr                                                                              64 hr                               ______________________________________                                        38       --           --        0.0  0.1  0.3                                   39 β-PMCD 56 2.6 3.7 0.0                                                 40 β-PMCD 62 2.9 4.2 2.5                                                 41 β-PMCD 68 2.8 3.3 2.5                                                 42 β-PMCD 69 2.9 0.0 2.2                                                 43 β-PMCD 74 2.9 1.8 2.6                                                 44 β-DMCD 67 3.2 3.1 2.1                                                 45 β-DMCD + TMCD 78 2.6 2.2 4.7                                          46 β-DMCD + TMCD 89 0.0 2.3 --                                           47 β-TMCD 100  0.0 0.1 0.2                                             ______________________________________                                         PMCD: partially methylated cyclodextrin                                       DMCD: 2,6di-O-methyl-cyclodextrin                                             TMCD: 2,4,6tri-O-methyl-cyclodextrin                                     

EXAMPLES 48-51 Preparation of 17,25-dihydroxycholesterol (Alteration inthe Substrate Concentration)

Fifty ml of the conversion culture medium described in Examples 29-37was placed in a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20minutes. This medium was inoculated with 1 ml of the seed culturesolution prepared as in Examples 29-37 and shaking culture was carriedout at 220 rpm for 48 hours at 28° C. To this culture were addedcholesterol in the form of powder and 5.6 ml of a 1.5% aqueous solutionof partially methylated β-cyclodextrin (methylation rate 74%: MericanCorp.) which was sterilized beforehand, and the cultivation continuedfor further 40 hours. By the same method as Examples 29-37,25-hydroxycholesterol in the resulting culture solution was quantified.The conversion rates into 17,25-dihydroxycholesterol from cholesterolwere summarized in Table 7.

                  TABLE 7                                                         ______________________________________                                        Example                                                                              Concentration                                                                              Concentration of                                                                             Conversion                                   No. of CHO added DiOH--CHO produced rate (%)                                ______________________________________                                        48     0.15 mg/ml   28 μg/ml    18.7                                         49 0.30 mg/ml 16 μg/ml 5.3                                                 50 0.60 mg/ml  6 μg/ml 1.0                                                 51 1.20 mg/ml  0 μg/ml 0.0                                               ______________________________________                                         CHO: Cholesterol                                                              DiOH--CHO: 17,25dihydroxycholesterol                                     

EXAMPLES 52 Preparation of 17,25-dihydroxycholesterol

A seed culture solution was prepared as in Examples 1-9.

Then, 1.5 l of a conversion culture medium consisting of 2.0% ofglucose, 0.2% of yeast extract, 0.5% of peptone, 1.0% of soybean meal,0.5% of corn steep liquor, 0.04% of potassium secondary phosphate, 0.04%of sodium chloride, 0.2% of calcium carbonate and 0.05% of Silicon KM75(defoamer: Shin-Etsu Chemical Co., Ltd.) (pH 7.4) was placed in a 3 lmini-jar and sterilized by heating at 120° C. for 20 minutes. Thismedium was inoculated with 30 ml of the seed culture solution preparedabove and cultivation was carried out for 48 hours at the temperature of28° C., at the agitation of 400 rpm and at the aeration of 1.0 vvm. Tothis culture, 450 mg of cholesterol dissolved in 15 ml of ethanol wasadded, 4 ml of a 10% aqueous solution of Silicon KM75 (defoamer:Shin-Etsu Chemical Co., Ltd.) and 30 ml of a 7.5% aqueous solution ofpartially methylated β-cyclodextrin (methylation rate 74%: MericanCorp.) were further added, and the cultivation continued for further 91hours.

Following the addition of Pearlite (filter aid: Toko Pearlite IND.) at aconcentration of 3%, the culture solution thus obtained was filtrated togive 1.2 l of filtrate. This filtrate was passed through a 100 mlAmberlite XAD-8 (Rohm & Haas) column to adsorb the product. Afterwashing this column with 200 ml of 20% aqueous methanol, the product waseluted with 500 ml of 90% aqueous methanol. After concentrating theeluate under reduced pressure to remove methanol, about 100 ml of theresidual aqueous layer was extracted twice with 50 ml of ethyl acetate.The resulting ethyl acetate layer was dried over anhydrous sodiumsulfate and concentrated to dryness.

After applying this residue to a 100 ml Silica gel (trade name: Silicagel 60, Merck) column, eluting with 400 ml of chloroform-methanol(100:1) and concentrating the eluate under reduced pressure to removethe solvent, the residue was further subjected to preparative thin layerchromatography (trade name: Silica gel 60 Art11798, Merck) and developedwith chloroform-methanol (20:1). The portion corresponding to25,26-dihydroxycholesterol (Rf=0.26) was scraped off, extracted withchloroform-methanol (1:1) and concentrated to dryness, giving 10.2 mg of25,26-dihydroxycholesterol in the form of white powder.

The physical and chemical properties of 25,26-dihydroxycholesterolprovided by the present invention were shown below:

(1) Appearance: white powder

(2) Melting point: 185-191° C.

(3) Molecular formula: C₂₇ H₄₆ O₃

(4) FAB mass spectrum (negative ion, matrix NBA): m/z=417 (M-1)

(5) ¹ H-NMR spectrum (400 MHz, CDCl₃): Main absorptions were as follows:

δTMS (ppm): 0.69 (3H, s), 0.93 (3H, d, J=6.6 Hz), 1.01 (3H, s), 1.14(3H, s), 3.37 (1H, d, J=11.0 Hz), 3.43 (1H, d, J=11.0 Hz), 3.50 (1H, m),5.34 (1H, m)

(6) ¹³ C-NMR spectrum (100 MHz, CDCl₃): Absorptions were as follows:

δTMS (ppm): 11.9 (q), 18.7 (q), 19.4 (q), 20.2 (t), 21.1 (t), 23.3 (q),24.3 (t), 28.2 (t), 31.7 (t), 31.9 (t+d), 35.7 (d), 36.5 (s+t), 37.3(t), 39.2 (t), 39.8 (t), 42.3 (t), 42.3 (t), 50.1 (d), 56.1 (d), 56.8(d) 70.0 (t), 71.8 (d), 73.0 (s), 121.7 (d), 140.8 (s)

EXAMPLES 53-61 Preparation of 25,26-dihydroxycholesterol (Effect of theAdditives)

A seed culture solution was prepared as in Examples 1-9.

Fifty ml of a conversion culture medium consisting of 2.0% of glucose,0.2% of yeast extract, 0.5% of peptone, 1.0% of soybean meal, 0.5% ofcorn steep liquor, 0.04% of potassium secondary phosphate, 0.04% ofsodium chloride and 0.2% of calcium carbonate (pH 7.4) was then placedin a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20 minutes.This medium was inoculated with 1 ml of the seed culture solutionprepared above and shaking culture was carried out at 220 rpm at 28° C.Fifteen mg of the substrate cholesterol and each compound which is shownin Table 8 were added 48 hours later, and the cultivation continued forfurther 72 hours.

The substrate and the like were added in the following way: Fifteen mgof cholesterol in the powder form was added in Example 53. For Example54, 150 mg of cholesterol was suspended in 4 ml of ethanol, dispersed bysonication, and 0.4 ml of the suspension was added. In Example 55, 150mg of cholesterol was suspended in a mixture of 1 ml of Tween 80(surfactant: Sigma) and 3 ml of methanol, dispersed by sonication, and0.4 ml of the suspension was added. In Examples 56, 57 and 58, therewere added 15 mg of cholesterol, 1 ml of Tween 80 and 5.6 ml of a 1.5%aqueous solution of each cyclodextrin which was sterilized beforehand(121° C., 20 minutes). In Examples 59, 60 and 61, there were added 15 mgof cholesterol, 1 ml of Tween 80 and a 1.5% aqueous solution ofpartially methylated β-cyclodextrin (methylation rate 74%: MercianCorp.), which was sterilized beforehand (121° C., 20 minutes), at eachdetermined concentration.

Two ml of the resulting culture solution was collected into acentrifugation tube with a stopper, to which 0.5 ml of ethyl acetate wasadded, and stirring for 30 minutes and further centrifugation at 3000rpm for 15 minutes separated off the ethyl acetate layer. Five μl ofthis was spotted onto a TLC plate (Silica gel 60 F₂₅₄ : Merck),developed with chloroform:methanol=10:1 and stained with sulfuric acid.The spot indicating the same Rf value (0.26) as the standard product inExample 1 was scanned by Chromatoscanner (CS-920: Shimadzu Corp.),25,26-dihydroxycholesterol being quantified. The conversion rates into25,26-dihydroxycholesterol from cholesterol were summarized in Table 8.

                  TABLE 8                                                         ______________________________________                                                             Concentration                                              Example Coexisting of coexisting Conversion                                   No. substance substance (%) rate (%)                                        ______________________________________                                        53       --          --         0.1                                             54 Ethanol 0.80 0.2                                                           55 Tween 80 0.20 0.1                                                          56 α-CD 0.15 0.3                                                        57 β-CD 0.15 0.2                                                         58 γ-CD 0.15 0.2                                                        59 β-PMCD 0.05 1.8                                                       60 β-PMCD 0.10 2.4                                                       61 β-PMCD 0.15 3.0                                                     ______________________________________                                         CD: cyclodextrin                                                              CD: cyclodextrin                                                              CD: cyclodextrin                                                              PMCD: partially methylated cyclodextrin                                  

EXAMPLES 62-71 Preparation of 25,26-dihydroxycholesterol (Effect of theReaction Time)

Fifty ml of the conversion medium described in Examples 53-61 was placedin a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20 minutes.This medium was inoculated with 1 ml of the seed culture solutionprepared as in Examples 53-61 and shaking culture was carried out at 220rpm for 48 hours at 28° C. To this culture, 15 mg of cholesterol in theform of powder and 1 ml of Tween 80 were added, a 1.5% aqueous solutionof each partially methylated β-cyclodextrin which was sterilizedbeforehand was further added so as to be a final concentration of 0.15%,and the cultivation continued. At 16, 40 and 64 hours after the additionof cholesterol, 25,26-dihydroxycholesterol in the culture solution wasquantified by the same method as Examples 53-61. The conversion ratesinto 25,26-dihydroxycholesterol from cholesterol were summarized inTable 9. In Examples 69 and 70, the mixture of2,6-di-O-methyl-β-cyclodextrin and 2,4,6-tri-O-methyl-β-cyclodextrinwere used at the ratios of 2:1 and 1:2, respectively.

                  TABLE 9                                                         ______________________________________                                        Example  Coexisting   Methylation                                                                             Conversion rate (%)                           No.      substance    rate (%)  16 hr                                                                              40 hr                                                                              64 hr                               ______________________________________                                        62       --           --        0.0  0.1  0.2                                   63 β-PMCD 56 3.3 2.5 0.0                                                 64 β-PMCD 62 2.8 2.3 2.3                                                 65 β-PMCD 68 3.3 3.0 3.2                                                 66 β-PMCD 69 3.9 1.0 1.7                                                 67 β-PMCD 74 2.2 2.5 2.6                                                 68 β-DMCD 67 3.5 4.1 3.7                                                 69 β-DMCD + TMCD 78 2.5 3.1 3.4                                          70 β-DMCD + TMCD 89 0.0 1.6 --                                           71 β-TMCD 100  0.0 0.1 0.2                                             ______________________________________                                         PMCD: partially methylated cyclodextrin                                       DMCD: 2,6di-O-methyl-cyclodextrin                                             TMCD: 2,4,6tri-O-methyl-cyclodextrin                                     

EXAMPLES 72-75 Preparation of 25,26-dihydroxycholesterol (Effect of theSubstrate Concentration)

Fifty ml of the conversion culture medium described in Examples 53-61was placed in a 250 ml Erlenmeyer flask and autoclaved at 120° C. for 20minutes. This medium was inoculated with 1 ml of the seed culturesolution prepared as in Examples 53-61 and shaking culture was carriedout at 220 rpm for 48 hours at 28° C. To this culture were addedcholesterol in the form of powder and 5.6 ml of a 1.5% aqueous solutionof partially methylated β-cyclodextrin (methylation rate 74%: MercianCorp.) which was sterilized beforehand, and the cultivation continuedfor further 40 hours. By the same method as Examples 53-61,25,26-dihydroxycholesterol in the culture solution obtained wasquantified. The conversion rates into 25,26-dihydroxycholesterol fromcholesterol were summarized in Table 10.

                  TABLE 10                                                        ______________________________________                                        Example                                                                              Concentration                                                                              Concentration of                                                                             Conversion                                   No. of CHO added DiOH--CHO produced rate (%)                                ______________________________________                                        72     0.15 mg/ml   30 μg/ml    20.0                                         73 0.30 mg/ml 18 μg/ml 6.0                                                 74 0.60 mg/ml  7 μg/ml 1.2                                                 75 1.20 mg/ml  0 μg/ml 0.0                                               ______________________________________                                         CHO: Cholesterol                                                              DiOH--CHO: 25,26dihydroxycholesterol                                     

EXAMPLES 76-88

A seed culture solution and a conversion culture medium were preparedsimilarly to those in Examples 1-9, except that a strain used wasreplaced with each strain which is shown in Table 11, and partiallymethylated β-cyclodextrin was added so as to be a final concentration of1.0%.

The concentration of cholesterol added was made to be 300 μg/ml and theculture was carried out at the pH and the reaction time which are shownin Table 11 below. The measurements of the substrate cholesterol and thehydroxylated cholesterols obtained were carried out in accordance withthose in the above Examples. The results were summarized in Table 11.

                                      TABLE 11                                    __________________________________________________________________________                 Reaction                     Conver-                               Example  time  Residual 25(OH) 17, 25(OH) 25, 26(OH) sion rate                No. Microorganism (hr) pH CHO --CHO --CHO --CHO (%)                         __________________________________________________________________________    76   Streptomyces                                                                          18   8.21                                                                             168.00                                                                             15.90                                                                             0.00  0.00  5.3                                   (Com- roseosporus 46 8.57 0.00 0.00 0.00 0.00 0.0                             parison)  68 8.83 0.00 22.60 0.00 0.00 7.5                                    77 FERM BP-5544 18 7.56 14.20 250.00 0.00 0.00 83.3                             48 7.58 0.00 290.00 23.10 28.70 96.7                                          68 7.97 0.00 269.00 22.10 39.30 89.7                                        78 FERM BP-2307 18 7.56 15.80 261.00 0.00 0.00 87.0                             46 7.56 0.00 289.00 21.80 25.00 96.3                                          68 7.90 0.00 240.00 19.00 23.10 80.0                                        79 Amycolate 18 7.40 111.00 200.00 0.00 0.00 66.7                              autotrophica 46 7.38 15.20 274.00 0.00 19.70 91.3                             ATCC 33796 68 7.97 0.00 246.00 0.00 14.00 82.0                               80 Sphingomonas 18 7.06 265.00 0.00 0.00 0.00 0.0                              IFO 15500 46 7.07 316.00 26.90 3.83 0.00 9.0                                   68 7.05 259.00 0.00 0.00 0.00 0.0                                           81 Sphingomonas 18 4.49 246.00 0.00 0.00 0.00 0.0                              IFO 13935 46 4.51 302.00 22.30 0.00 0.00 7.4                                   68 4.47 239.00 19.40 0.00 0.00 6.5                                          82 Sphingomonas 18 4.92 228.00 0.00 0.00 0.00 0.0                              IFO 15100 46 4.76 302.00 13.40 0.00 0.00 4.5                                   68 4.68 244.00 28.30 0.00 0.00 9.4                                          83 Sphingomonas 18 4.92 225.00 0.00 0.00 0.00 0.0                              IFO 15102 46 4.90 296.00 16.10 0.00 0.00 5.4                                   68 4.87 219.00 0.00 0.00 0.00 0.0                                           84 Sphingomonas 18 7.11 210.00 0.00 0.00 0.00 0.0                              IFO 15099 46 5.38 274.00 0.00 0.00 0.00 0.0                                    68 5.05 167.00 10.90 0.00 0.00 3.6                                          85 Sphingomonas 18 7.13 245.00 0.00 0.00 0.00 0.0                              IFO 12533 46 7.76 286.00 0.00 0.00 0.00 0.0                                    68 8.07 203.00 23.40 4.03 8.20 7.8                                          86 Sphingomonas 18 5.09 233.00 0.00 0.00 0.00 0.0                              IFO 13937 46 5.13 260.00 23.00 0.00 0.00 7.7                                   68 5.13 199.00 0.00 0.00 0.00 0.0                                           87 Sphingomonas 18 7.41 230.00 0.00 0.00 0.00 0.0                              IFO 15033 46 7.66 269.00 0.00 0.00 0.00 0.0                                    68 7.74 210.00 23.70 0.00 0.00 7.9                                          88 Sphingomonas 18 8.30 232.00 0.00 0.00 0.00 0.0                              IFO 15098 46 8.33 270.00 13.70 0.00 0.00 4.6                                   68 8.41 239.00 0.00 0.00 0.00 0.0                                         __________________________________________________________________________

wherein

CHO: Cholesterol

25(OH)--CHO: 25-hydroxycholesterol

17,25(OH)--CHO: 17,25-dihydroxycholesterol

25,26(OH)--CHO: 25,26-dihydroxycholesterol

Industrial Applicability

According to the present invention, hydroxylated cholesterols including25-hydroxycholesterol can be efficiently prepared, and17,25-dihydroxycholesterol and 25,26-dihydroxycholesterol are providedas new hydroxylated cholesterols. These compounds are useful, forexample, as intermediates for the preparation of vitamin D compounds andwould be applicable in manufacturing industries for medicaments.

What is claimed is:
 1. A method for preparing a hydroxylated cholesterolhaving the formula (II): ##STR4## in which R¹ is a hydroxyl radical, andR² and R³ are a hydroxyl radical and a hydrogen atom respectively, or ahydrogen atom and a hydroxyl radical respectively, by biologicalconversion of cholesterol having the formula (I): ##STR5## which methodcomprises: (A) incubating cholesterol having the formula (I) with astrain of Amycolata or a preparation thereof which is able tobiologically convert said cholesterol into the hydroxylated cholesterolhaving the formula (II), in an atmosphere containing oxygen, to producean incubation-treated solution, in which (1) the hydroxylatedcholesterol is either a compound having the formula (II) wherein R¹ andR² are a hydroxyl radical, and R³ is a hydrogen atom, or a compoundhaving the formula (II) wherein R¹ and R³ are a hydroxyl radical, and R²is a hydrogen atom, and the strain of Amycolata is Amycolata saturneaFERM BP-5544, or (2) the hydroxylated cholesterol is a compound havingthe formula (II) wherein R¹ and R³ are a hydroxyl radical, and R² is ahydrogen atom, and the strain of Amycolata is Amycolata autotrophicaATCC 33796; and(B) recovering the hydroxylated cholesterol from theincubation-treated solution.
 2. The method according to claim 1, whereinincubation is carried out in the further presence of methylatedcyclodextrins.
 3. A method for preparing a dihydroxycholesterol havingthe formula (II-b): ##STR6## in which R² and R³ are a hydroxyl radicaland a hydrogen atom respectively, or a hydrogen atom and a hydroxylradical respectivelyby biological conversion of 25-hydroxycholesterolhaving the formula (II-a): ##STR7## which method comprises: (A)incubating 25-hydroxycholesterol having the formula (II-a) withAmycolata saturnea FERM BP-5544, or a preparation thereof which is ableto biologically convert said 25-hydroxycholesterol into thedihydroxycholesterol having the formula (II-b), in an atmospherecontaining oxygen, to produce an incubation-treated solution, and (B)recovering the dihydroxycholesterol from the incubation-treatedsolution.
 4. The method according to claim 3, wherein incubation iscarried out in the further presence of methylated cyclodextrins.